The generation of asymmetry during development and the polarization of differentiated cells require asymmetric distributions of cytoplasmic proteins. Messenger RNA localization provides an important mechanism for generating protein asymmetries by targeting the synthesis of such proteins to specific subcellular domains. Polarized cellular functions like motility in fibroblasts and synaptic plasticity in dendrites, asymmetric division of budding yeast, and embryonic axis formation in Xenopus and Drosophila all require proteins that are synthesized from localized mRNAs. The proposed research aims to elucidate mechanisms of mRNA localization used to generate polarity during development. These studies focus on nanos mRNA, whose localization to the posterior pole of the Drosophila embryo is essential for production of a Nanos protein gradient that patterns the anterior-posterior body axis. Work during the previous grant period has shown that localization of nanos is also required for germ cell function and for the proper development of peripheral neurons. The proposed studies combine molecular, biochemical, and genetic approaches to determine how mRNAs like nanos are specifically recognized by localization factors and how these RNA-protein interactions result in transport of mRNAs to their target destinations and maintain them there. Live imaging now permits investigation of mechanisms used by nanos mRNA for localization in different cellular contexts. Aim 1 focuses on biochemical analysis of nanos localization factors isolated during the previous funding period as well as purification of additional factors using an affinity purification strategy. Aim 2 takes a complementary approach toward identification of proteins involved in nanos mRNA localization through a sensitized genetic screen. Aims 3 and 4 take advantage of a system for in vivo fluorescent labeling of mRNAs to follow the dynamics of nanos localization in live embryos and in neurons. In addition, a new system that permits visualization of two mRNAs simultaneously will expand our understanding of how multiple mRNA localization pathways intersect. Public Health Relevance: Messenger RNA localization produces localized protein distributions required for embryonic axis formation and asymmetric cell division during development and for polarized cellular functions like motility in fibroblasts and synaptic plasticity in dendrites. Altered levels of proteins that bind to messenger RNAs and direct their localization have been associated with a variety of cancers and the tumorigenicity of these proteins may reflect their roles in localizing mRNAs for cell fate specification, cell polarity, and migration. The proposed studies will shed light on mechanisms by which RNA-protein interactions provide the highly selective control of basic cellular processes needed for development, growth, and differentiation and how the disruption of these processes may lead to diseases like cancer or neurological dysfunction.